MDR1 shRNA对人脑胶质瘤干细胞多药耐药性实验研究

Regulatory effect of shRNA targeting MDR1 on multidrug resistance of human brain glioma stem cells

目的构建多药耐药基因(MDR1)的短发卡RNA表达质粒,并检测其对胶质瘤干细胞药物敏感性的作用。方法培养脑胶质瘤干细胞;根据MDR1的DNA序列设计shRNA,用Siport xp-1脂质体法转染胶质瘤干细胞。分别采用定量聚合酶链反应(PCR)和Western blot检测转染前后MDR1 mRNA和蛋白表达情况;使用活细胞计数试剂盒(CCK-8)对转染后细胞进行药物敏感性试验,评价shRNA对多药耐药性的逆转作用。结果成功构建MDR1 shRNA表达质粒,转染组MDR1 mRNA表达水平有所下降(P<0.05),转染5d组下降最明显;RT-PCR结果显示MDR1 mRNA水平降低,第3、5、7d抑制率分别为78.46%±14.17%,82.02%±11.87%,79.5%±13.27%。Western blot结果显示:shRNA转染组P-糖蛋白(P-gp)的表达降低,第3、5、7d抑制率分别为58.1%±6.5%,39.5%±5.2%,45.8%±8.6%;而药敏实验显示:阿霉素、长春新碱对转染MDR1 shRNA的胶质瘤干细胞的半数抑制浓度(IC50)均有不同程度降低,且出现凋亡峰(P<0.05)。结论MDR1短发卡RNA可在转录后水平对多药耐药进行调节,下调MDR1基因表达,提高药物敏感性,诱导细胞凋亡。

Objective To explore the regulatory effect of small hairpin RNA （shRNA） targeting muhidrug resistant protein 1 （ MDR1 ） on chemosensitivity of human brain glioma stem cells. Methods Human brain glioblastoma stem cells were cultured; MDR1 shRNA was constructed according to GenBank MDR1 sequence and were cloned to the retrovirus-delivered vectors, shRNA expressing plasmids were delivered into human brain glioblastoma stem cells by using Siport xp-1. The mRNA and protein expressions of MDR1 were confirmed by reverse transcription polymerase chain reaction （RT-PCR） and Western blot respectively. Drug sensitivity assay was performed in the transfected cells. Results The plasmid expressing shRNA against MDR1 was successfully constructed and the given plasmids were amplified and verified by sequencing; human brain glioma stem cells proliferated exponentially after MDR1 shRNA transfection. RT-PCR showed MDR1 mRNA was significantly reduced. RT-PCR showed inhibition ratio of MDR1 was 78. d6% ±14. 17%, 82.02% ± 11.87% and 79.51%± 13.27% at day 3, 5, and 7, respectively. Western blot analysis demonstrated that P-gp expression was reduced at different times. Western Blot showed inhibition ratio of MDR1 was 58.1% ±6.5%, 39.5%±5.2% and 45.8% ±8.6% at day 3, 5, and 7, respectively. The 50% inhibitory concentrations （IC50） of doxorubicin and vincristine to human brain glioma stem ceils were decreased markedly. Conclusion MDRI shRNA may modulate multidrug resistance through down-regulating the expression of MDR1 gene and enhancing the chemosensitivity of gliomas.